Alessandra Panattoni

Advanced methods are necessary to improve the detection of flavescence dorée (FD), one of the most relevant grapevine (Vitis vinifera) diseases in Europe, caused by flavescence dorée phytoplasma (FDp). Detection is commonly carried out visually by agronomists/winegrowers, and is time-consuming and error-prone. The present study demonstrated that full-range hyperspectral data (i.e., 400 to 2,400 nm) collected at leaf level can be used as a tool to rapidly and nondestructively detect FD infection directly in the field. Focusing on a Sangiovese (red grape) vineyard of Tuscany (Central Italy), heavily affected by FD (incidence higher than 75%), we showed that the proposed hyperspectral approach is capable of (i) detecting FDp infection, even before the occurrence of leaf symptoms (accuracy >70%); (ii) discriminating FD-induced leaf symptoms, even between asymptomatic and lightly symptomatic leaves (accuracy 80%); and (iii) elucidating the complex physiological responses of grapevines to FDp infection, with changes in leaf parameters estimated from spectra suggesting that the disease not only impaired early-season photosynthetic efficiency but also accelerated leaf senescence, potentially impacting grapevine productivity and grape quality. Although the hyperspectral approach proposed here is not intended to replace traditional diagnostic methods (molecular analyses), it could serve as a valuable tool to support the monitoring of plants affected by FD and may represent a crucial advancement in FD management. Further and broader studies including vineyards less challenged by FD and with other grape varieties (e.g., white ones, showing leaf yellowing instead of reddening) are encouraged.

Flavescence dorée (FD) is a major grapevine disease in Europe, despite the quarantine status of its causal agent [FD phytoplasma (FDp)] and the mandatory monitoring and vector control practices. As alarming FD epidemic outbreaks continue to appear in Tuscany (Central Italy), a 4-year survey was carried out in the main wine-growing areas of the region, where FD presence was investigated in both primary and secondary FDp hosts and vectors, i.e., Vitis vinifera (VV), Clematis vitalba (CV), Alnus glutinosa (AG), Scaphoideus titanus (ST), and Dictyophara europaea (DE). This work i) confirmed FD diffusion in almost the whole of Tuscany and even with an increased occurrence rate (approximately 50% of the samples tested positive); ii) highlighted a complex FDp ecology also in the Tuscan vineyard agroecosystem, as FDp was reported not only in VV and ST but also in secondary vectors (DE) and hosts (AG and CV); iii) reported nine FDp strains (three of which were novel) belonging to all the three methionine aminopeptidase (map) clusters, i.e., map-FD1, map-FD2, and map-FD3, with the map-FD3/M51 genotype mostly reported in not only VV but also DE and CV; and iv) further confirmed a complex FDp ecology in the vineyard agroecosystem, also by phylogenetic analyses carried out at both Italian and European levels, which also showed some relations between the Tuscan FDp strains and those reported in the Balkan and the French scenarios. We believe that the outcomes reported here will be useful in preventing and controlling the spread of harmful FD.

Sharka, caused by plum pox virus (PPV), is by far the most harmful disease of stone fruits (genus Prunus). Although Italy is a major producer of stone fruits, and sharka has spread in most Italian regions since its first identification in the 1970s, information on the diffusion of the disease in Italy results still scattered and incomplete. To address this knowledge gap, the present review aimed to firstly provide a historical overview of sharka in Italy, summarizing outcomes on the disease achieved since the first identification of its causal agent PPV. Data retrieved from a variety of sources (i.e., published literature, official reports and personal communications) showed that (i) sharka spread across almost the whole Italian peninsula as only Liguria, Umbria and Aosta Valley regions officially result disease-free; (ii) the regions with higher rates of infected districts are not always those more dedicated to the production of stone fruits; (iii) only the three major PPV strains causing sharka, i.e., PPV-Dideron (D), PPV-Marcus (M), and PPV-Recombinant (Rec), have been reported in Italy; and (iv) only apricot, peach and plum (i.e., widespread and economically relevant species) are hosts of PPV, with different scenarios among regions. A focus on Tuscany (Central Italy) showed that, (i) sharka has been reported since the 1990s, but extensive PPV monitoring campaigns were run from 2013 to 2021, especially in nurseries; (ii) higher number of symptomatic and PPV positive samples were recorded in farms, mostly in the Arezzo and Florence districts; and (iii) only PPV-M and PPV-Rec strains seem still present. We believe that the present overview will benefit and encourage both researchers for future investigations on sharka and farmers that have to manage the disease.

Wisteria vein mosaic virus (WVMV) is a potyvirus infecting Wisteria spp. worldwide, making these largely used ornamental plants unattractive and even unsalable. In 2021, nine Wisteria sinensis plants in Sarzana (Liguria, Italy) showing WVMV symptoms like vein mosaic with irregular patterns, mottling, deformation and twisting margin on leaves were reported. This work describes research on symptomatic and asymptomatic leaves to confirm the identity of the virus infection. All plants tested resulted positive to WVMV. Sequencing of NIb/CP genomic region and comparison on GenBank revealed the presence of eight new genetic variants named Sar 5–12. The eight nucleotide sequences alignments revealed identity ranging between 87.13 and 99.85%. Negative selection (dN/dS < 1) was detected suggesting well adaptation in the area here examined and stability in population. Through a phylogenetic tree, WVMV isolates were grouped in four clades with high bootstrap values, two of which included the eight Italian variants here identified. Since the late 1950 and 1960s, the present study represents the first additional report of WVMV in Italy, as well as its first molecular characterization ever. Further research is required to strengthen our understanding of the movement and transmission of WVMV. Examining how virus infected plants can disseminate this pathogen could help forestall potential risks that neighbouring species may face in their environments.

The present study focused on trees of Tuscany autochthonous plum cv. Coscia di Monaca in order to evaluate the presence of viruses and elucidate the physiological and biochemical responses to virus infections under real field conditions. Among the several investigated viruses, plums tested positive only to plum pox virus (PPV) and plum bark necrosis steam pitting associated virus (PBNSPaV), occurring as both singular and co-infections. This is the first report of PBNSPaV in a Tuscany orchard. Furthermore, the present study not only confirmed the detrimental effects of PPV on the carbon dioxide assimilation rate due to both stomatal limitations and mesophyll impairments, but also showed that although PBNSPaV did not induce such photosynthetic impairments when occurring as singular infection, it enhanced this damaging effect when present as a co-infection with PPV, as confirmed by a severe decrease in the chlorophyll content. Infection-specific responses in terms of accessory pigments (i.e., carotenoids and xanthophylls), as well as sugars and organic acids, were also reported, these being likely related to photoprotective mechanisms and osmotic regulations under virus-induced oxidative stress. Overall, the results here presented represent an important step to fill knowledge gaps about the interaction of plant viruses and autochthonous Prunus cultivars.

“Bois noir” disease associated with ‘Candidatus Phytoplasma solani’ seriously compromises the production and survival of grapevines (Vitis vinifera L.) in Europe. Understanding the plant response to phytoplasmas should help to improve disease control strategies. Using a combined metabolomic and transcriptomic analysis, this work, therefore, investigated the phytoplasma–grapevine interaction in red cultivar Sangiovese in a vineyard over four seasonal growth stages (from late spring to late summer), comparing leaves from healthy and infected grapevines (symptomatic and symptomless). We found an accumulation of both conjugate and free salicylic acids (SAs) in the leaves of ‘Ca. P. solani’-positive plants from early stages of infection, when plants are still asymptomatic. A strong accumulation of gentisic acid (GA) associated with symptoms progression was found for the first time. A detailed analysis of phenylpropanoids revealed a significant accumulation of hydroxycinnamic acids, flavonols, flavan 3-ols, and anthocyanin cyanidin 3-O-glucoside, which are extensively studied due to their involvement in the plant response to various pathogens. Metabolomic data corroborated by gene expression analysis indicated that phenylpropanoid biosynthetic and salicylic acid-responsive genes were upregulated in ‘Ca. P. solani-positive plants compared to -negative ones during the observed period.

Plum pox virus (PPV) a potyvirus in the family Potyviridae, is the etiologic agent of sharka, the worldwide most important and detrimental viral disease of stone fruits (genus Prunus). Although PPV was identified in Italy already in the 1970s and it has been largely reported in almost all Italian regions, a broad investigation and genetic characterization of PPV isolates was lacking in most of them, including Tuscany (Central Italy). To address this knowledge gap, in 2020 and 2021, field surveys were carried out in 80 commercial Tuscany orchards and a total of 690 leaf samples were collected from different Prunus species. PPV was identified in 25 and 12 peach and plum samples, respectively (more than 5% of tested samples), whereas no positive samples were reported in apricot. Eighteen of the 37 PPV positive samples showed mixed infections with other viruses and viroids, mostly Prunus necrotic ringspot virus in plum and peach latent mosaic viroid in peach. Molecular typing of PPV infected samples generated NIb/CP amplicons corresponding to PPV-Marcus (M) or PPV-Recombinant (Rec) strains. Furthermore, starting from the identification of eight PPV nucleotide sequences (among which five and two new PPV-M and PPV-Rec isolates, respectively), this study firstly identified the PPV-Mb subgroup in Italy, which was even prevalent than PPV-Ma. Finally, PPV-Rec isolates resulted phylogenetically close to Italian and Turkish isolates previously detected. Overall, the results here presented represent an important step to fill knowledge gaps about PPV in Tuscany, and we believe it may encourage other similar research to achieve more accurate data on PPV populations at both national and international levels.

Grapevine “bois noir”, related to the presence of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), represents a serious threat in several vine-growing areas worldwide. In surveys conducted over two years, mild and/or moderate symptoms and lower pathogen titer were mainly associated with ‘Ca. P. solani’ strains harboring a secY gene sequence variant (secY52), whereas severe symptoms and higher titer were mainly observed in grapevines infected by phytoplasma strains carrying any one of another four variants. A comparison of amino acid sequences of the protein SecY of ‘Ca. P. solani’ strains revealed the presence of conservative and semi-conservative substitutions. The deduced three-dimensional (3D) protein analysis unveiled that one semi-conservative substitution identified in the sequence variant secY52 is responsible for a structural disordered region that probably confers a flexibility for binding to distinct molecular complexes. In fact, the other analyzed variants show an organized structure and the 3D in silico prediction allowed the identification of β-sheets. Thus, differences in symptom severity and pathogen concentration observed in grapevines infected by ‘Ca. P. solani’ strains carrying distinct secY gene sequence variants suggest a possible relationship between SecY protein structure and phytoplasma strain virulence.

Grapevine Bois noir (BN) is associated with infection by “Candidatus Phytoplasma solani” (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the CaPsol strains was carried out by analysis of genes encoding 16S rRNA and translation elongation factor EF-Tu, as well as eight other previously uncharacterized genomic fragments. Strains of tuf-type a and b were found to be differentially distributed in the examined geographic regions in correlation with the prevalence of nettle and bindweed. Two sequence variants were identified in each of the four genomic segments harboring hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS-tyrS-csdB, respectively. Fifteen CaPsol lineages were identified based on distinct combinations of sequence variations within these genetic loci. Each CaPsol lineage exhibited a unique collective restriction fragment length polymorphism (RFLP) pattern and differed from each other in geographic distribution, probably in relation to the diverse ecological complexity of vineyards and their surroundings. This RFLP-based typing method could be a useful tool for investigating the ecology of CaPsol and the epidemiology of its associated diseases. Phylogenetic analyses highlighted that the sequence variants of the gene hlyC, which encodes a hemolysin III-like protein, separated into two clusters consistent with the separation of two distinct lineages on the basis of tufB gene sequences. Alignments of deduced full protein sequences of elongation factor-Tu (tufB gene) and hemolysin III-like protein (hlyC gene) revealed the presence of critical amino acid substitutions distinguishing CaPsol strains of tuf-type a and b. Findings from the present study provide new insights into the genetic diversity and ecology of CaPsol populations in vineyards.

Bois noir (BN), associated with ‘Candidatus Phytoplasma solani’ (CaPsol), is the most widespread disease of the grapevine yellows complex worldwide. In this work, BN epidemiology was investigated in a case study vineyard where an unusual CaPsol strain, previously detected only in other host plants, was found to be prevalent in grapevine. Experimental activities included: symptom observation; sampling of symptomatic vines, Auchenorrhyncha specimens, and weeds; molecular detection and typing of CaPsol strains; statistical analyses for determining possible relationships between CaPsol relative concentration, strain type, and symptom severity. Among insects, Reptalus quinquecostatus was the most abundant and was found to be highly infected by CaPsol, while Hyalesthes obsoletus, the main CaPsol vector, was not caught. Moreover, R. quinquecostatus harbored CaPsol strains carrying uniquely the stamp sequence variant St10, also identified as prevalent in vines and in the majority of weeds, and all the secY variants identified in the vineyard. Statistical analyses revealed that CaPsol strains carrying the St10 variant are not associated with severe symptoms, suggesting their possible moderate virulence. Based on such evidence, a new BN epidemiological pattern related to these CaPsol strains and involving grapevine, R. quinquecostatus, and/or weeds is proposed. Furthermore, the possible presence of other players (vectors and weeds) involved in CaPsol transmission to grapevines was highlighted.